Among agents identified as potential weapons under CDC category A, or high priority agents, is botulinum toxin (BoNT). BoNT poses a major bioweapon threat because of its extreme potency and lethality, its ease of production and transport, as well as the need for prolonged intensive care among affected persons. The goal of this proposal is to develop immune-based therapeutics to counter the use of BoNT as a weapon of bioterrorism. Since BoNT cannot cross intact skin, when used as a bioweapon, it is expected that BoNT would gain entry via epithelial cells of the gastrointestinal (GI), respiratory tract or both. Experiments are proposed to determine what sequences are required for BoNT transport by human epithelial cells. We will first determine the minimal toxin fragment required for uptake across polarized human epithelial cells. Both binding and transcytosis will be measured. Site-directed mutation will be used to further map the site on the toxin required for binding, transcytosis or both. Polarized epithelial cell lines originating from both the lung and the GI tract will be used to determine if access across different epithelia requires the same toxin domain. Monoclonal antibodies (mAbs) will be produced to the domain (or sub-domain) responsible for epithelial binding and transcytosis. The mAbs will be tested for their ability to inhibit binding and/or transcytosis. Both the intracellular trafficking pathway and the receptor(s) on the epithelium used for transcytosis will be identified. If a protein functions in the receptor, it will be cloned. The role of antibody isotype and valence in the inhibition of BoNT transport across the epithelium will be determined. Variable regions from mAbs will be cloned and expressed as IgA, which is polymeric and hence exhibits enhanced avidity, and as secretory IgA (slgA) with covalently attached secretory piece, which is also polymeric but is more resistant to proteases. The role of effector function activation and valence in the context of IgG will also be investigated. Efforts will be made to produce an antibody-based protein that can gain access to the cytosol of the neuron and neutralize intracellular BoNT. mAbs effective in neutralizing the catalytic activity of BoNT will be produced. We will attempt to deliver these into the cytoplasm of neurons either by genetically fusing them to antibodies that recognize surface molecules of neurons and are endocytosed or by replacing the catalytic domain of BoNT with a scFv version of the neutralizing mAb.